Detection and changes in levels of testosterone during spermatogenesis in the freshwater planarian Bdellocephala brunnea

It was reported recently that vertebrate-type steroids exist and control reproduction in several groups of invertebrates, including molluscs. Sexually reproductive freshwater planarians of the species Bdellocephala brunnea have a limited breeding season in their natural habitat. This phenomenon suggests that some endogenous reproductive hormones might play a role in vivo. However, to date, sex steroids such as androgen, estrogen, and progesterone have not been found in planarians. The goal of the present study was to determine whether androgen is present in sexual planarians such as B. brunnea. The presence of testosterone was detected by high-pressure liquid chromatography and, in sexually reproductive individuals in which no seminal vesicles were visible, the level of testosterone was about twice than that in individuals with visible seminal vesicles. An enzyme-linked immunosorbent assay revealed that the levels of testosterone during terminal spermatogenesis were three times higher than during the spermatocyte-building phase. Our results indicate that sexually reproductive freshwater planarians such as B. brunnea might have vertebrate-type steroids and show variation in testosterone levels during spermatogenesis.     

An electron-microscopic study of syncytium formation during early embryonic development of the freshwater planarian Bdellocephala brunnea

To substantiate the assumption that the egg cell and blastomeres in planarian embryos influence surrounding yolk cells to form a syncytium, embryos at 1- to 8-cell stages were examined by electron microscopy. Within special areas of the endoplasmic reticulum both in the egg cell and in the blastomeres, a large number of vacuoles of various sizes formed and then disappeared at least four times over the period from egg-laying through the 8-cell stage as if their contents were being secreted. These activities diminished markedly at the 8-cell stage. Yolk cells surrounding the egg cell and blastomeres were aggregated in close contact with one another in a small clump shortly after egg-laying, and then, late in the 4-cell stage, became fused, forming a syncytium. The correlation between release of vacuoles by the egg cell and blastomeres and the formation of a syncytium by the yolk cells indicate that the cell fusion could be induced by a factor contained in the vacuoles.     

野葛块根脱分化与再分化相关因子研究

采用正交试验、单因子试验和植物组织培养方法,探讨几种因子对野葛块根组织脱分化与再分化的影响。结果表明,野葛块根愈伤组织诱导的最佳培养基为MS+NAA 0.5mg/L+6-BA 1.0mg/L+2,4-D 2.0mg/L,暗培养更有利于愈伤组织的诱导;野葛块根愈伤组织的最佳出芽培养基为MS+NAA 0.5mg/L+6-BA 3.0mg/L或MS+NAA 0.5mg/L+KT 2.0mg/L,光照培养更有利于愈伤组织芽的再分化;野葛块根愈伤组织再生芽生根最佳培养基为MS+NAA 0.5mg/L+PP₃₃₃ 3.0mg/L+蔗糖30g/L;PP₃₃₃ 3.0mg/L和蛭石:珍珠岩(2:1)基质能显著提高再生苗的移栽成活率。     

Formation of malformed pharynx and neoplasia in the planarian Bdellocephala brunnea following treatment with a carcinogen

The effect of benzo(a)pyrene on regeneration of the pharynx in Bdellocephala brunnea Ijima et Kaburaki was studied by inserting a 5-µg crystal of this carcinogen into the pharyngeal cavity of worms from which the pharynx had been extirpated 2 d previously. Multiple pharynges formed in the regenerates, and neoplasia was induced in a few cases. The malformed pharynges appeared as protrusions overlapping the anterior part of the main pharynx. Electron microscopy of the pharynx in normal, intact worms shows eight layers comprising inner and outer ciliated epithelia, muscle, and glands.     

Switching from bone marrow-derived neurons to epithelial cells through dedifferentiation and translineage redifferentiation

While the ability of stem cells to switch lineages has been suggested, the route(s) through which this may happen is unclear. To date, the best characterized adult stem cell population considered to possess transdifferentiation capacity is BM-MSCs (bone marrow mesenchymal stem cells). We investigated whether BM-MSCs that had terminally differentiated into the neural or epithelial lineage could be induced to transdifferentiate into the other phenotype in vitro. Our results reveal that neuronal phenotypic cells derived from adult rat bone marrow cells can be switched to epithelial phenotypic cells, or vice versa, by culture manipulation allowing the differentiated cells to go through, first, dedifferentiation and then redifferentiation to another phenotype. Direct transdifferentiation from differentiated neuronal or epithelial phenotype to the other differentiated phenotype cannot be observed even when appropriate culture conditions are provided. Thus, dedifferentiation appears to be a prerequisite for changing fate and differentiating into a different lineage from a differentiated cell population.     

On the origin of neoblasts in freshwater planarians (Turbellaria)

Experiments on 1) regeneration of the cave-adapted planarian, Sphalloplana zeschi, 2) induction of sexuality in an asexual strain of Dugesia japonica japonica by feeding, and 3) culture of dissociated planarian cells, show that neoblasts originate from intestinal cells, i.e. phagocytic cells and granular clubs.     

Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians.     

In vitro Propagation,Dedifferentiation and redifferentiation of Marchantia polymorpha L.

The gemma and gametophyte of Marchantia #olymorpha were propagated in vitro. Dedifferentiation and redifferentiation as well as the media used and cultural conditions reguired were described. Since the differentiation of bryophytes was very difficult, it was necessary to culture the tissue through initiation of partial dedifferentiation on MS agar medium supplemented with 1 mg/1 2,4-D and 3% sucrose, and then subsequently the tissue was transplanted onto 1/2 Knop agar medium with addition of 4–8 mg/1 2,4-D, 0.25–0.5 mg/1 BA and Fe salt of MS medium. The formed calli were visual but still contained rhizoid, in this stage. The small calli finally were subcultured in white agar medium supplemented with mixture of pyruvic acid, citric acid and fumaric acid (5 mmol/1); 1 mg/1 2,4-D and 4% glucose. They could be differentiated thoroughly into normal tissue. The time of the total process for differentiation requied as long as 10 months. The redifferentiation and regeneration of thalli were far easier than those of higher plants even if they were transplanted onto MS phytohormone-free medium.     

小麦成熟胚脱分化过程CDPKs基因的差异表达分析

用Affymetrix小麦基因芯片检测小麦成熟胚脱分化过程中CDPKs基因的表达变化的结果表明,在检测到的16个CDPKs基因中,有15个基因在小麦成熟胚脱分化过程中发生了有意义变化,其中上调表达基因有12个,上调2-7．5倍,下调表达基因有2个,下调3娟．1倍,在不同时点既有上调又有下调的1个。根据已知CDPKs生物功能扣它们表达变化趋势以及这些变化趋势与脱分化过程中的吻合程度分析结果,推测CPK2A、CPK2B．CPK6和Os12g0169800基因参与了小麦成熟胚脱分化过程的启动和愈伤组织的形成。     

红豆杉脱分化过程中的遗传和表观遗传变异

采用扩增片段长度多态性（AFLP）和甲基化敏感扩增多态性（MSAP）技术分析红豆杉脱分化前后基因组DNA和DNA甲基化状态的变化。选用32个AFLP引物组合从红豆杉植株及其愈伤组织分别扩增出1834个片段,无多态性片段产生。这说明红豆杉植株在诱导形成愈伤组织的过程中基因组DNA保持高度的遗传稳定性。另用32个MSAP引物组合从红豆杉植株及其愈伤组织分别扩增出1197个片段,总扩增位点的甲基化水平由脱分化前的12．4％上升为16．2％,表明红豆杉在脱分化过程中的某些位点发生了甲基化。红豆杉脱分化前后的DNA甲基化模式也存在较大差异,说明DNA甲基化对愈伤组织形成有调控作用。     

Dedifferentiation derived cells exhibit phenotypic and functional characteristics of epidermal stem cells

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell‐like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation‐derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c‐nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and β₁‐integrin emerged at the wound‐neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and β₁‐integrin⁺ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10⁺ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation‐derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, β₁‐integrin, Oct4 and Nanog in dedifferentiation‐derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation‐derived cells demonstrated: (1) the numbers of colonies consisting of 5–10 cells and greater than 10 cells were increased 5.9‐fold and 6.7‐fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 × 10⁵ cells versus 1 × 10¹² cells) were all significantly increased and (4) dedifferentiation‐derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation‐derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.     

Dedifferentiation of chloroplasts in interspecific and homospecific protoplast fusion products

Summary Interspecific and homospecific protoplast fusion products (Vicia narbonensis +V. hajastana,V. hajastana+V. hajastana) regenerated cell walls and divided when culturedin vitro for a period of 7 days. While most organelles appeared typical of actively growing plant cells, chloroplasts exhibited structural changes which were interpreted as dedifferentiation.     

The anatomy of Microplana termitophaga from Zimbabwe and Kenya: confirmation of the subfamily and genus

Microplana termitophaga has been observed feeding on termites in Harare, Zimbabwe. In small specimens the pharynx is about two-thirds of the way along the body, but in large specimens the pharynx is only one quarter of the way along the body. Details of the subepidermal musculature of specimens from Kenya and Zimbabwe are described which confirm the previously uncertain assignment of the species to the subfamily Microplaninae. Sexually mature specimens are found towards the end of the wet season in Zimbabwe. The anatomy of the reproductive system is described. The gonopore is about two-thirds of the way along the body in mature specimens, but sexual maturity seems not to be related simply to size. There is a genito-intestinal duct which confirms that the species is of the genus Microplana.     

Development of the reproductive apparatus of the land planarian Rhynchodemus sylvaticus (Turbellaria: Tricladida) and its significance for classification in the genus

Seven specimens of Rhynchodemus sylvaticus (Leidy) collected from a variety of localities in the US and having variously developed copulatory organs are believed to represent stages in the development of the copulatory apparatus. Four specimens were juveniles with under-developed male components, one specimen had a well-developed female atrium and small male component, and two specimens were mature with a male organ twice the size of the female part. In early stages, the male component had sperm ducts, seminal vesicle, and narrow atrium; more mature stages had a considerable elongated atrium with thick folds in its muscularized wall, a massive muscular bulbus; and a sigmoid ejaculatory duct opening into the large bulbar cavity. Morphological features of mature male copulatory organs in all species of the genus Rhynchodemus are basically similar whereas external body features (color and number of dorsal stripes) of these same species differ.